Instrument and methods for reducing the thickness of a vitrified aqueous specimen

Abstract

The present invention relates to an instrument for preparation of frozen specimens, and a method for such preparation; in particular, applicable to specimens for cryomicroscopy. A method for reducing the thickness of a vitrified aqueous specimen (101) is disclosed.

Description

[0001] INSTRUMENT AND METHODS FOR REDUCING THE THICKNESS OF A VITRIFIED AQUEOUS SPECIMEN

[0002] Related Applications

[0003] The present application is related to, and claims priority to and the benefit of GB 231 1680.9 filed on 28 July 2023 (28.07.2023), the contents of which are incorporated by reference in their entirety.

[0004] Field of the Invention

[0005] The present invention relates to an instrument for preparation of frozen specimens, and a method for such preparation; in particular, applicable to specimens for cryomicroscopy.

[0006] Background

[0007] Whilst electron cryomicroscopy (cryo-EM) has recently become a powerful method for determining the structure of many proteins and complexes that were difficult or even intractable by X-ray crystallography, the production of a frozen specimen suitable for high resolution data collection remains a severe bottleneck.

[0008] On a perfect, well-prepared grid (cryo-EM grids being a well known way of mounting a specimen for analysis), containing a uniformly thin layer of highly concentrated and pure protein suspended in vitreous ice, enough data can be collected in two hours on a modern cryomicroscope to solve a 3 A structure. Yet despite this fact and the vastly increased capacity for cryo-EM now available, structure determination often takes years and dozens of microscope days. Nearly all of this time is spent preparing and examining grids where the specimen has somehow been damaged or destroyed during the grid preparation process. This is a trial-and-error process that can take months or years, particularly for small and challenging proteins like G-protein coupled receptors (GPCRs) of great interest for therapeutics.

[0009] Many approaches have been investigated to address the air-water interface problem (whereby particles which have interacted with the air-water interface may lose their integrity or become highly orientated and therefore interfere with or even prevent accurate structure determination), including the use of functionalised support films to provide an alternative interface or the addition of surfactants to the specimens. None of these approaches proved to be a universal solution, since in all cases, there remain interactions between the specimen and the modified surface of the thin film, which may still be detrimental to the structure of the molecules present.

[0010] A second major problem in biological microscopy is the thinning of cells or tissue specimens, which are generally far too thick for imaging with electron microscopes. Currently this is most commonly done with high energy (1 -30 keV) focused ion beam (FIB) instruments that cut small sections or lamellae, from a large biological specimen that was frozen by plunge or high pressure freezing. This cryo-FIB milling process is slow, inherently serial and fraught with difficulty. Material can be removed at rates of at best ~1013atoms / second and only from small regions on the micrometer length scale. Further time is required to polish the surfaces of the lamella that are produced since the high energy beam leaves behind a severely damaged layer whose thickness is proportional to the energy of the ions being used, about 1 nm / kV. Since charged particle optics limit the precision with which an ion beam can be focused at low energies, and of course FIB methods require focused beams to work, this means there is a lower limit to the thickness of damage layers created that is several nanometres and in most cases more like 10s of nanometres thick.

[0011] For 30 keV beams, which are most commonly used to mitigate the effects of charging and non-uniform milling caused by non-uniform composition in the specimen, the damage layer is 30-50 nm thick rendering most of a thin lamella (typically 100 - 200 nm in total thickness) destroyed even before imaging and increasing the background signal for the undamaged central regions of the specimen.

[0012] The present invention has been devised in light of the above considerations.

[0013] Summary of the Invention

[0014] The present inventors have developed methods and an instrument to use low-energy ions or neutral atoms to mill (etch) vitrified aqueous specimens, in order to make thin, damage free films suitable as specimens for, for example, electron cryomicroscopy (cryoEM). The methods and instrument can be applied to the thinning of any frozen biological specimen, such as cells, tissues, organelles, and purified proteins, to the sub-1 pm thicknesses required for imaging with high resolution cryoEM. Unlike current technologies that use high-energy focused ion beams, which leave damaged regions on the surfaces of the subsequent specimens, this method uses only diffuse beams of ions at low energy (<1000 eV) to etch away the frozen water and solutes without damaging the molecules below, even those just below the etched surface.

[0015] Conditions for selective removal of the aqueous specimen versus the support and other surfaces are established herein. Since the ion ‘beam’ is diffuse (many orders of magnitude larger than the electron beams used to subsequently image the specimens created), many regions of multiple specimens can be thinned at the same time, and specific regions can suitably be selected using a mask placed close (for example, within one mean free path of the ion) to the surface to be milled. By controlling the distance between the mask and the surface, gradient profiles in milling rates across an individual region, an entire specimen support or several supports can also be achieved. During specific etch procedures and depending on the ion species selected, lower temperatures are preferred but also need to be high enough to prevent condensation of the corresponding gases on to the surface. Thus an optimal range of temperatures and pressures for a given ion species is established herein. A range of different ion / gas mixtures have been tested and are compared. In some instances, conditions may be optimised for fast removal rates of bulk material. In other instances, conditions may be optimised for fine surface polishing of small amounts from an already thin specimen.

[0016] An instrument for achieving this etching or milling is also described herein. Accordingly, a first aspect of the invention provides a method of reducing the thickness of a vitrified aqueous specimen, the method comprising exposing the specimen to broad (not strongly focused) beam of ions of energy <1000 eV while the specimen is at a temperature <150 K and in the presence of <10-6Torr of water vapour partial pressure.

[0017] This can be used to create a specimen that is precisely the correct dimensions for imaging using an electron microscope, and that does not contain any particles that have interacted with the interfaces of the specimen during formation (in particular the air-water interface). In this way the particles imaged are those that were freely floating in solution immediately prior to the freezing, rather than those adhered to the surface of the water layer, as those surfaces will be completely removed. This can address, for example, two major problems in single-particle cryoEM: (1 ) preferred specimen orientations, and (2) the partial or complete denaturation of the specimen at the hydrophobic air-water interface, as well as other problems discussed herein.

[0018] In some embodiments the ions have an energy of <500 eV, preferably <300 eV, and more preferably <100 eV. This can enable yet further fine control and selectivity of etching and hence thickness. The ion energy can be freely controlled down to 1 eV or even a fraction of an eV.

[0019] In some embodiments the ions have an energy of >1 eV, preferably >2 eV, more preferably >3 eV, more preferably >4 eV, more preferably >5 eV, more preferably >10 eV. This can give an appreciable speed of etching. At the relevant temperature water contaminants from the chamber atmosphere may deposit on the specimen and hence will need to be etched away. The etching speed is ideally enough to overcome this deposition rate as well as provide the desired specimen thinning.

[0020] Suitably the specimen is exposed while at a temperature of <120 K. This ensures the vitreous nature of the specimen is maintained. In some embodiments the specimen is exposed while at a temperature of <100K.

[0021] In some embodiments the specimen is exposed while at a temperature of >20 K, >50K or >78K, which can help minimise condensation of gases onto the specimen surface.

[0022] In some embodiments the specimen is exposed to the ion beam in the presence of < 10-7Torr or < 10-6Torr of water vapour partial pressure. Most preferably the water vapour partial pressure is < 10-9Torr or < 10-10Torr, which can help lead to faster milling and a flatter resulting surface. In some embodiments the ions are generated from helium, nitrogen, argon or neon plasma.

[0023] In some embodiments the ions are generated from a mixture of helium, nitrogen, neon and argon plasma.

[0024] In some embodiments the ions are generated from at least one of helium, nitrogen, argon or neon plasma and at least one of oxygen or hydrogen.

[0025] In some embodiments the specimen is exposed at a pressure of 1 mTorr to 250 mTorr, 1 mTorr to 150 mTorr, or preferably 5 to 100 mTorr.

[0026] The vitrified aqueous specimen may suitably be held on a cryoEM specimen support, suitable for imaging in a transmission electron microscope.

[0027] A second aspect of the invention provides a method of preparing a vitrified aqueous specimen, comprising the steps of

[0028] (a) mounting or forming the vitrified aqueous specimen on a stage, the specimen having an initial thickness;

[0029] (b) conducting a method of reducing the thickness of the vitrified aqueous specimen according to the first aspect, until the thickness of the specimen is reduced from the initial thickness to a target thickness which is smaller than the initial thickness.

[0030] Suitably the target thickness is <1 pm, preferably 10-100 nm. Such thicknesses are appropriate for electron microscopy.

[0031] In some embodiments the initial thickness, which is greater than the target thickness, is >20 nm, preferably >30 nm. It may be thicker still, even into the 10s of pm (for example, up to 10 pm, up to 50 pm, or up to 99 pm).

[0032] A third aspect of the invention provides an apparatus for reducing the thickness of a vitrified aqueous specimen, comprising: an ion source which is a plasma generator or an ion beam generator; a process chamber connected to the ion source; and a stage, positioned to hold a specimen within the process chamber; a pressure control means connected to the process chamber, operable to reduce the pressure in the process chamber; and a temperature control means connected to the stage, operable to reduce and maintain the specimen temperature therein; wherein the process chamber is configured to maintain and withstand a specimen temperature of <150 K, preferably <120 K, a pressure of <250 mTorr, preferably < 150 mTorr, even more preferably <100 mTorr and a water vapour partial pressure of < 10-6Torr, and wherein the ion source is configured to generate ions of energy <1000 eV.

[0033] It is apparent that the apparatus of the third aspect may be for use in conducting the method of the first aspect or the method of the second aspect.

[0034] In some embodiments the apparatus further comprises a shuttle for placing on the stage and having a mounting area for mounting the specimen, and a shutter on the shuttle which is moveable from a first position, in which the mounting area is exposed, and a second position, in which the mounting area is at least partially concealed. This allows for control of exposure of the specimen to the process chamber, as well as protection of the specimen during movement around the apparatus (e.g. during loading or mounting).

[0035] There may also be provided a mask, for example as part of the shuttle or as a separate part of the apparatus, to selectively pattern areas of the specimen. The mask may for example be placed in the mounting area when a specimen is present, either in contact with the specimen or close to it.

[0036] The apparatus may also comprise a load lock chamber which is attached to the process chamber by an air-tight seal and separated from it by a valve such as a gate valve, and a pump connected to the load lock chamber and operable to reduce the presume in the load lock chamber. The specimen can be loaded into such a chamber, which is then evacuated, before being transported through the gate valve into the process chamber for treatment. This greatly reduces the chance of contaminants such as water entering into the process chamber.

[0037] In some embodiments, the apparatus further comprises one or more gas supplies connected to the ion source, for supplying one or more processing gases to the ion source, and one or more cold traps, each corresponding to a respective gas supply and being positioned in the flow path between the gas supply and the ion source. By having each or any processing gas pass through a cold trap before entering the ion source (i.e. before being used for ion generation), the presence of contaminants such as water can be minimised. In some embodiments, contaminants such as water can be reduced using a high surface area zeolite cold trap. Alternatively, contaminants such as water can be reduced using a desiccant, or by other chemical reaction.

[0038] The methods and instrument disclosed here allows one to achieve thickness removal rates over large areas, for example over the area of multiple 3 mm diameter electron microscopy support grids, with the removal rate independent of the etched area. For example, multiple cells on multiple cryoEM grids can all be thinned down to a thickness amenable to cryoEM simultaneously, during a single exposure to the etching process, instead of cutting sections through the cells one by one as is currently done in cryoFIB milling. This is because the proposed method uses flood (diffuse), rather than focused, ion beams for etching. Thus, the proposed methods can address two major problems of cryoFIB milling: (1 ) the damage layer on the surface of the specimens thinned by high-energy ion beams, and (2) the inherently serial nature and slow speed of the focused ion beam milling process, which requires rastering the focused beam around the area that needs to be removed. By the present invention, the need for high resolution imaging during milling, a vibration free stage, and complicated and expensive focusing optics for the beam is removed.

[0039] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0040] Summary of the Figures

[0041] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0042] Figure 1 is a diagrammatic representation of an example etching carried out according to the present invention.

[0043] Figure 2A shows a schematic representation of the removal of thin vitreous ice with undercutting below a gold support foil during milling; Figure 2B shows a transmission electron micrograph (TEM) using bright field image F20 TEM at 200 keV.

[0044] Figure 3 shows bright field TEM images, imaged at 100 keV using a direct electron detector, to demonstrate the complete removal of thin vitreous ice (approximately 30 nm initial thickness) after exposure to 20 minutes of neon plasma at 210 mTorr and 60 Kelvin with applied specimen bias of -40 V, achieving an etch rate of > 1 nm / minute and no detectable removal of the supporting gold foil. Figure 3A shows before the plasma etching; Figure 3B after. Figure 4 shows bright field TEM images, imaged at 100 keV using a direct electron detector, to demonstrate the preservation of biological specimen structure before and after etching. Figure 4A shows a specimen after exposure to 1 minute of neon plasma at 210 mTorr and 60 Kelvin with applied specimen bias of -40 V. Proteins may be distributed on both surfaces of the specimen and inside the thin film of ice. Plasma was applied from one side of the specimen which will have removed a portion of the proteins from one of the surfaces. Consequently, a portion of the particles will have been damaged. Figure 4B and Figure 4C are the results of two-dimensional classification from 50,000 particles micrographs; the results of two-dimensional averaging and classification of the particles (DPS) resulted in two classes of particle, demonstrating that particles are preserved through the etching process. For comparison, all the molecules within 30-60 nm of the surface are damaged or destroyed in FIB milling with 30 keV ions.

[0045] Figure 5 shows a similar demonstration as in Figures 3 and 4 but with a Ribosome specimen. 37,000 particles were picked from micrographs acquired from the specimen after plasma application from a single side. After two-dimensional averaging and classification, 2856 particles were selected into three classes. De novo three-dimensional reconstruction generated a three-dimensional map at a resolution of 5.2 A. Figure 5A shows the two-dimensional classes, Figure 5B the three-dimensional reconstruction and Figure 5C the Fourier shell correlation to demonstrate the resolution calculated from two independently determined half-maps.

[0046] Figure 6 shows SEM micrographs of a grid frozen with a thick layer of amorphous ice, before (Figure 6A) and after (Figure 6B) plasma etching. In Figure 6A the whole area of the grid is coated with amorphous ice, with thickness equal to that of the grid bars (10 pm) or more. After the exposure to the plasma, in Figure 6B all the ice is removed, exposing the metal grid bars.

[0047] Figure 7 shows the use of a mask (schematically shown in Figure 7A) to selectively thin the specimen. Figure 7B shows the result of an experiment demonstrating the masked etching of a region of a grid as schematically shown in Figure 7A (in a composite low-magnification transmission micrograph of the grid after the exposure to the plasma). Figure 7C is a high-magnification transmission electron micrograph from the region indicated with the arrow in Figure 7B and shows particles preserved within the remaining vitrified ice layer.

[0048] Figure 8 is a graph of measured vitreous ice sputtering rate as a function of ion energy.

[0049] Figure 9 is a plot of current measured through the specimen stage as a function of applied bias during exposure to different plasmas. In the negative bias region, the positive ions are attracted to the surface. The opposite occurs for the positive potential - the positive ions are repelled from the surfaces and the current is due to electrons (and possible some negative ions) hitting the surfaces of the stage and specimen.

[0050] Figure 10A shows a diagram of an instrument for low-energy plasma etching of cryogenic specimens according to the present invention. Figures 10B, C and D show photographs of the plasma tube and main chamber with viewport during operation, the cryotransfer system of the instrument which allows transfer of specimen shuttles from the specimen loader pot into a UHV (ultra-high vacuum) load lock (top viewport), where the specimen is docked onto a liquid nitrogen cooled stage before it is loaded to the main chamber, and the cold stage inside the main chamber with the specimen shuttle on it, respectively.

[0051] Figure 11 is a plot showing the heat load on the stage measured for different gases at 90 K and a pressure of 0.1 Torr.

[0052] Figure 12 is a diagram of a setup for optical feedback control of specimen thickness during plasma etching. The light beam is transmitted through the specimen at a particular wavelength and is modulated in intensity at a particular frequency to allow detection using lock-in techniques even with the bright background created by the light emitted by the plasma. The transmission is then used to measure the amount of material remaining and thus monitor etching in real-time and control the final thickness of the specimen. The plasma can also be turned off intermittently to make a more accurate measurement of the progress of the etching.

[0053] Detailed Description of the Invention

[0054] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0055] Methods

[0056] The present methods are based on thinning a specimen by exposure to ions of energy <1000 eV at a temperature of <150 K and in the presence of < 106Torr of water vapour partial pressure.

[0057] The low energy of the ions ensures that the thickness of the damage layer on the surface of the etched specimen is less than 1 nm. An energy of <1000 eV is used, but lower energies may be preferred to yet further reduce the thickness of the damage layer. For example, the ions may preferably be of energy <500 eV, <300 eV, <200 eV, <100 eV, <50 eV, <20 eV, <10 eV or even less than <5 eV.

[0058] On the other hand, a certain level of ion energy may be necessary in order for etching to proceed at an appreciable rate. Thus, in some embodiments the ions may suitably have an energy of >1 eV, preferably >2 eV, >3 eV, >4 eV, >5 eV, >10 eV or >20 eV.

[0059] The treatment temperature is <150 K to retain the vitreous nature of the aqueous specimen. Ideally the temperature is <120 K, at which temperature de-vitrification may begin to proceed. Similarly, a temperature >20 K may suitably be used to give a superior treatment rate and to reduce the chance of condensation of the processing gas (or any other gas present) on the surface of the specimen.

[0060] The specimen is generally kept at such temperatures throughout the process.

[0061] Therefore, ideally, the specimen (and for example the stage upon which it sits) is cooled by a system able to provide these temperatures and account for any thermal load exerted by the process gas and plasma onto the specimen during the process. The present inventors have measured the thermal load exerted by some example process gases and their plasmas in exemplary process pressure ranges, and fit these to a theoretical model based on kinetic heat exchange between the warm gas molecules and the cold stage. Results are shown in Figure 11. They have found that, within these operating pressures of the instrument, a cooling power of ~10 Watts is required to prevent the specimen stage from heating during the process, and the exact amount of cooling power required depends on the process gas. Most traditional cryo holders employed, for example in cryoTEMs, do not deliver sufficient power (~1 W) to be used in these process conditions.

[0062] The present ions may be generated from an ion source such as a plasma generator or other ion beam generator.

[0063] Here it is important to note that while the present application refers to an “ion beam”, this does not mean that the “beam” is necessarily focused, of small cross section, or similar. This terminology is simply used to refers to an ion ‘flow’, as is usual in the art.

[0064] In the present invention, the ions are not part of a focused beam; that is, they are unfocussed or diffuse. Their movement may be controlled, for example to impart acceleration in a given direction such as towards a specimen to be treated, by application of an electrical bias.

[0065] A bias applied to the specimen, shuttle (or other specimen holder), or stage allows acceleration of the ions, leading to higher rate of sputtering but less selective removal. Monitoring the progression of etching while varying the bias can be used to discern when an area of the metal support foil is first exposed to the plasma. Final polishing can then take place at a lower bias which is more selective for sputtering of ice, so that the specimen holder is not itself eroded.

[0066] The bias may suitably be, for example, from -1000V to 0 to +1000V. More preferably the bias may be - 300V to +100V, for example -200V to +50V, -200V to 0V or -100V to 0V, or -50V to 0V or +50V to 0V (i.e. a negative bias may be preferable in some cases).

[0067] The ions themselves may suitably be generated from a low-energy plasma, pulsed plasma or ion beam, itself formed from a process gas. Suitable sources (i.e. suitable process gases) are, for example, argon, carbon dioxide, carbon monoxide, ethylene, fluorine, helium, krypton, methane, neon, nitrogen, nitrogen trifluoride, oxygen and xenon.

[0068] Most preferably, one of helium, nitrogen, argon or neon gas is used, sometimes in combination with each other or with the addition of some oxygen to enhance the hydrocarbon etching potential.

[0069] The low energy ions remove material from the vitreous specimen (including water, protein, carbohydrates, lipids, etc), gradually thinning it down to the desired target thickness, where the final thickness is determined by the duration of exposure to a particular plasma. A specimen such as a 3 mm grid containing a vitreous biological specimen for electron microscopy (or even larger) can be exposed and thinned down in a single exposure during this process.

[0070] By use of unfocused ions, a much larger area can be concurrently treated. Therefore, although a given addressed location may be etched more slowly due to the low ion energy as compared to an FIB method, since the removal occurs over a vastly greater area (rather than only in the area of focus) the throughput is greatly enhanced.

[0071] The pressure used for the present process is, suitably, low. The ideal, minimum and maximum pressure and temperature may be determined based on, for example, the temperature at which the process gas condenses at given pressure.

[0072] In the present methods, a pressure of the treatment atmosphere (i.e. of the chamber containing the ion beam / plasma and the specimen to be processed) may suitably be, for example, 1 mTorr to 250 mTorr, for example 1 mTorr to 150 mTorr, for example 5 to 100 mTorr. However, a pressure up to 1 Torr or even 10 Torr may be useful in some cases where very high fluxes are desired. In the present methods, it is required that the specimen is exposed to the ion beam in the presence of < 10-6Torr of water vapour partial pressure. Most preferably the water vapour partial pressure is < 10-9Torr or < 10-10Torr. Lower partial pressures of water vapour contamination will lead to faster milling and as such a flatter resulting surface. Preferable conditions for a range of possible process gases are set out in Table 1 below.

[0073] Table 1

[0074] Illustrative Embodiments

[0075] Testing has shown that application of low-energy plasmas is effective for etching of thin vitreous specimens, prepared for cryoEM. Various plasma compositions, including argon, oxygen, nitrogen, neon and helium have been shown to remove amorphous water and biological material embedded in it.

[0076] Etching rates of approximately 1 nm / minute have been demonstrated, resulting in surfaces sufficiently smooth for imaging by cryoEM and preservation of the embedded protein within the thinned layer.

[0077] A simplified schematic illustration of an embodiment of the present invention is shown in Figure 1. A standard cryoEM support (gold foil 105, with holes therein, formed on a support grid comprising support bars 106 (also, for example, gold) and having a vitreous aqueous specimen 101 immobilized thereon) is shown in the plasma 108 or ion beam described herein. A mask 107 ‘conceals’ part of the specimen 101 from the plasma 108 (left hand side of the diagram). Etching of the specimen 101 , reducing its thickness, occurs outside the masked region (right hand side of the diagram); it can be seen that the “etched region” 102 extends slightly under the masked area based on the mean free path of the plasma. It can also be seen that the etched region 102 increases slightly in thickness as the distance from the mask 107 increases. These effects are discussed in more detail below, regarding thickness control.

[0078] An enlarged portion of Figure 1 shows in more detail the specimen 101 on the foil 105; it has a free diffusion region 104 where the species to be imaged are held, and an interface 103 which contacts the atmosphere. The interface 103 is etched by the plasma 108, exposing the free diffusion region 104. Figure 2 illustrates the selective removal of a thin vitreous ice with undercutting below a gold support foil during milling, and the possibility of patterning ice using undercutting below a tight mask. A nitrogen plasma at 20 mTorr, 60 W was generated and the specimen was exposed for 2000 seconds. The estimated removal rate is 0.5 A / second in this condition. Figure 2A is a diagrammatic cross section of the embodiment; Figure 2B an SEM image of an experimental repeat. The undercut region can be seen as a clouded periphery around the bright etched region seen through the hole in the foil.

[0079] Figure 3 illustrates the complete removal of thin vitreous ice, after exposure to 20 minutes of neon plasma at 210 mTorr and 60 Kelvin stage temperature with an applied bias of -40 V. Since Initial thickness is approximately 30 nm, an etch rate of > 1 nm / minute for ice with no detectable removal of the supporting gold foil was achieved. Figure 3A shows before etching; Figure 3B shows after. It can be seen that, after etching, the thin ice is removed entirely and the thicker ice partially removed.

[0080] Figure 4 illustrates the preservation of a biological specimen within a thin vitreous ice layer after exposure to 1 minute of Neon plasma under the same conditions as above (210 mTorr, -40 V, 60K). Based on Figure 3, it is estimated that around 1 nm of material is removed from the top surface of the specimen. By two-dimensional averaging of the protein particles (DPS) in the remaining thin layer, it was found that the structure of the protein is preserved after the plasma etching (compare example micrograph Figure 4A and classes from two-dimensional averaging in Figures 4B and 4C).

[0081] Figure 5 illustrates the preservation of a biological specimen in thin vitreous ice layer after exposure to 60 minutes of neon plasma under the conditions 210 mTorr, -40 V applied bias and 65 Kelvin stage temperature. Two-dimensional averaging of the protein particles (70S Ribosome) in the remaining thin layer has been preserved after plasma etching as demonstrated by several two-dimensional classes Figure 5A and three-dimensional reconstruction to 5.2 A Figure 5B as determined by Fourier shell correlation shown in Figure 5C.

[0082] Thick frozen vitreous specimen can also be removed over a wide area using the present method, as illustrated in Figure 6. Specimens can be frozen as a thick amorphous layer by high pressure freezing, or by plunge freezing in presence of high concentration of protein or cryoprotectants, or any other method such as bringing the specimen into rapid contact with a cryogenically cooled, polished surface (slam freezing).

[0083] The method is capable of removal of a thicker vitreous frozen specimen layer from the total area of the microscopy support grid. A ten (10) micrometer layer of amorphous ice with suspended protein as seen in Figure 6A was totally removed as seen in Figure 6B, after plasma etching of the whole grid for 1 hour using nitrogen plasma at 20 mTorr, with a stage temperature between 90 K and 120 K.

[0084] Instrument

[0085] At its broadest, the present instrument or apparatus includes an ion source, a process chamber connected to the ion source; and a stage, positioned to hold a specimen within the process chamber. It typically also has a pressure control means connected to the process chamber, operable to reduce the pressure in the process chamber; and a temperature control means connected to the process chamber, operable to reduce the temperature therein.

[0086] The apparatus is generally constructed to be useful in the methods described herein. Accordingly, the process chamber is configured to maintain and withstand a temperature of <150 K, preferably <120 K, a pressure of <250 mTorr, preferably <100 mTorr and a water vapour partial pressure of < 10-6Torr, and the ion source is configured to generate ions of energy <1000 eV. The ion source is, as described herein, generally a plasma generator or other ion beam generator.

[0087] The ion source, which provides the low-energy ions used in the present invention, may be for example a radio frequency plasma generator.

[0088] The process chamber is for example an ultra-high vacuum reaction chamber with variable pumping capacity provided by the pressure control means (for example, a turbomolecular pump).

[0089] The chamber may be provided with one or more viewports, and / or equipment to measure plasma conditions and chamber conditions such as pressure gauges (Baraton, cold cathode gauge, Pirani gauge) or light transmittance equipment as described herein with reference to Figure 12 for monitoring specimen thickness.

[0090] The stage may ideally be electrically isolated, so that it can be addressed with an electrical bias as described herein to accelerate or retard ions to the stage and hence to the specimen.

[0091] Around the specimen stage (for example to surround the stage in all sides except for that which faces the ion source), a cryoshield may be provided to help to achieve higher cooling power, and further remove contaminants such as water vapour from the system. The cryoshield can prevent water vapour from reaching the specimen, for example, and hence prevent condensation of it onto the specimen. The cryoshield can also shield the stage from gas travelling at angles apart from the plasma direction.

[0092] The cryoshield can be cooled to a lower temperature than the stage to achieve greater trapping of, for example, water vapour, and can either be grounded to the outer chamber potential, or have a bias applied with electrical isolation in the same way as the main stage, but with the bias chosen to minimise erosion of the ice trapped on its surface. This will aid to lower the rate of deposition and redeposition of ice onto the surface in some plasma conditions.

[0093] As explained herein, control and minimisation of water contamination is a significant factor in the present invention. Water vapour present may be present in the ambient environment, which is present to an extent on all surfaces of the chamber, and may be present in trace amounts in the process gases.

[0094] To achieve thinning of the specimen, and hence to thin it using the plasma milling system, the removal rate of the specimen must be appreciably greater than the deposition rate of water vapour from the gases. While water may not be completely eliminated from the system, lower rates of water vapour contamination will lead to faster milling with flatter resulting surface. They are hence advantageous.

[0095] The process chamber may therefore suitably be constructed with features to minimise water contamination. That can be monitored by for example using a residual gas analyser, and optical spectroscopy during plasma processing. Such equipment may be connected to a viewport of the process chamber for information gathering.

[0096] The temperature control means primarily adjusts the temperature of the stage, as that is the region in which the specimen will be treated. However, it may be suitably powered to control the temperature throughout the process chamber.

[0097] During plasma processing, the insulating surface of the frozen specimen can become charged. This can suppress the influx of ions and reduce the etching rates if not controlled in some cases. It may also eventually act to attract ice crystals present in liquid nitrogen or in the dry air around the specimen to the surface of the specimen during transfer between vacuum systems, or during storage in liquid nitrogen. A charge neutralisation device in the process chamber can suitably be used as a source of electrons or ions with either positive or negative charge to neutralise this residual charge during or after plasma etching. Alternatively, the plasma system can be alternately used with a positive and negative bias to impinge electrons onto the surface in between cycles of etching with positive ions or vice-versa.

[0098] The present apparatus may comprise a shuttle for placing on the stage. The shuttle maybe a moveable item which holds the specimen itself, for example in a mounting area. That mounting area might be, for example, an aperture through the shuttle, across / over which the specimen itself may be mounted. As described herein, the specimen may be held on, for example, a cryoEM grid.

[0099] The shuttle can be moved around the apparatus to transport the specimen, for example from a loading hatch into the process chamber and onto the stage. In view of that, the shuttle may suitably be provided with means of protecting the specimen during movement. For example, the shuttle may have a shutter which can be moved over the mounting area to protect the specimen when loaded. The shutter, in the closed position, may together with the shuttle completely encase the specimen so that it is not exposed to potential damage. The shutter may seal the specimen from external atmosphere, protecting it from ingress of contaminant gases such as water vapour, for example during loading.

[0100] The shuttle may suitably be made from material with high thermal conductivity to ensure high cooling power to the specimen, such as pure copper with low oxygen content, or gold. The same is true of the stage.

[0101] Since copper and gold are the metals most susceptible to plasma sputtering, a coating can be used on the stage and on the internal surfaces of the process chamber to allow processing with higher energy ions whilst maintaining the necessary selectivity. Coating with a refractory metal can increase the resistance to plasma, such as with nickel, aluminium platinum, iridium, or platinum iridium alloy, tantalum, tungsten or by oxide, carbide or nitride, or coating by carbon, silicon or silicon oxide or nitride. Corrosion of these materials by the plasma can also be a concern which may enhance sputtering, such as formation of copper oxide by reaction with oxygen present in the vacuum at the specimen surface. Thin layers of these materials could be applied by electron beam evaporation, thermal evaporation or by electroplating. The best choice will be influenced by ease of fabrication, and could be optimised depending on the gas species of the plasma. The stage and shuttle may each have electrical connections, positioned to connect the shuttle and the stage electrically when the shuttle is placed on the stage. In this way, electrical properties of the shuttle can be analysed and electrical bias, as described herein, can be induced. For example, through electrical probing the shuttle temperature can be monitored.

[0102] There may also be provided one or more masks, as discussed herein, as part of the shuttle for controlling exposure of the specimen mounted in the mounting area during processing. The mask or masks may comprise, for example, a metal plate with one or more apertures therein. A mask can be positioned on or proximal to the specimen as described herein to control the area which is exposed to ions during processing and hence to control the pattern of etching.

[0103] The shuttle may be provided with multiple mounting areas, such that plural specimens can be held by a single shuttle. In this way throughput of the apparatus can be enhanced. Of course, in such embodiments each mounting area may for example have a respective shutter of the type discussed above, or respective mask / s. Alternatively, a single shutter and / or mask which acts on all specimens held by the shuttle may be provided. Such a single shutter may be operable to expose different specimens (i.e. different mounting areas) for different periods of time; for example, a staged retraction of the shutter, exposing a series of mounting areas in stages, may be permitted.

[0104] The present apparatus may suitably comprise one or more gas supplies, which deliver processing gas(es) to the ion source. For example, gas(es) for conversion to low energy plasma. Flow of such gases to the ion source may be controlled by respective mass flow controllers, MFCs. Hence the gas supply may be in fluid communication with the respective MFC, which is in turn in fluid communication with the ion source (e.g. plasma generator).

[0105] In order to minimise contamination, for example with water vapour, there may be a cold trap positioned in the flow path between the gas supply and the MFC (and / or, alternatively or additionally, in the flow path between the MFC and the ion source).

[0106] Commercially available high purity gases are at best 99.9999% pure and still may contain ppm levels of water vapour, or can become contaminated through leaks in the standard plumbing fittings. Preferably, for the flow path of the processing gas(es) vacuum compatible fittings are used, for example with shutoff attached to a regulator to allow testing of vacuum compatibility I leak rate into the system and elimination of leaks as far as possible. A fully metal sealed system is preferred to minimise leak rate, and the number of joints is ideally minimised in the gas inlet systems.

[0107] The use of a cold trap on the process gas inputs before the mass flow controller efficiently captures water vapour. Cold trapping water in the process gas lines can be achieved by, for example, passing a length of the flow tubing (which may be, for example, stainless steel) through liquid nitrogen. This direct cooling is applicable for gases which do not condense at the temperature of liquid nitrogen and the pressure of the MFC inlet (13.5 psi / 1 bar) such as nitrogen, neon, helium, hydrogen. This will remove water vapour to a level below 10-10Torr. For other gases, such as oxygen and argon, a heat transfer system with temperature control can be used over a length of tube. This can be vacuum isolated, and cooled using a nitrogen cooled cold finger or the like, with a braid attachment between a cold block attached to the steel tubing, which is heated, and a cold finger.

[0108] At process pressures, none of the gases used condense at liquid nitrogen temperature. An alternative or additional trap for water vapour can be applied after the gas(es) pass through the mass flow controller(s). Here the pressures are typically in the range 1 mTorr to 1 Torr, often in range 10 mTorr — 200 mTorr. This post-MFC cold trap can take the form of, for example, a cold finger in a separate vacuum system which is not exposed to the plasma, positioned between the mass flow controller(s) and the process chamber. This chamber can for example be isolated from the process chamber and pumped to ultra-high vacuum. This ‘gas dryer chamber’ may have a separate pressure gauge, burst disk and viewport. Gas enters from an inlet manifold, comes into contact with the liquid nitrogen cooled cold finger, and exits through an egress manifold, for example through a length of spiralled copper tube wrapped around the cold finger.

[0109] The present apparatus may also include a load lock chamber which is attached to the process chamber by an air-tight seal and separated from it by a gate valve, and a pump connected to the load lock chamber and operable to pump the atmosphere out of the load lock chamber to reduce the pressure therein.

[0110] The load lock chamber acts as an ante-chamber into which the specimen, for example loaded on the shuttle, can be introduced and conditioned before introduction into the process chamber for placing on the stage.

[0111] For example, the load lock chamber may be another ultra-high vacuum chamber with a stage, cooled by a temperature control means to temperatures of, for example, 90-330K. The chamber may be coupled to an air-free transfer system for moving a specimen from its storage holder, for example in liquid nitrogen or cold nitrogen gas, into the load lock chamber and from there into the processing chamber.

[0112] Such an additional antechamber can minimise the risk of contaminant ingress into the process chamber, and can allow the process chamber’s vacuum, for example, to be relatively unaffected by a loading / unloading operation the shuttle.

[0113] The load lock chamber stage may suitably be cooled to, for example, below 270K, for example below 150K and preferably below 120K, such that it can act as a cold trap for water vapour. Most preferably the temperature of the load lock chamber stage is below 150K, or suitably below 120K, whenever a specimen is present in it, in order to preserve the vitreous nature of the specimen.

[0114] For safety, the process chamber, the load lock and the gas dryer may each independently have independent pressure measurement, and burst discs.

[0115] The load lock chamber is suitably provided with handling means for moving the specimen within the load lock chamber. Suitably, those means allow movement of the specimen from load lock chamber to the process chamber and in particular to the stage. For example, there may be provided one or more handling arms which can be moved within the load lock chamber, and can extend into the process chamber to the stage. The load lock chamber may suitably have one or more viewports to allow observation of the interior.

[0116] An example apparatus of the present invention is schematically illustrated in Figure 10A; photographs of certain sections are in Figure 10B, Figure 10C and Figure 10D. It will be appreciated that features of this exemplary embodiment may be applied to other various embodiments where appropriate; in particular, whether certain features have no dependence on other features described in this particular embodiment.

[0117] With reference to Figure 10A, there is illustrated an apparatus of the present invention 1001 . It comprises an ion source which is a plasma generator 1002. In this embodiment, the plasma is generated in a glass (quartz) vacuum tube, surrounded by a conductive coil. The current in the coil is oscillated at a frequency of 13.6 MHz, and the resulting fields in the tube generate the plasma from process gases (Gas 1 , Gas 2, Gas 3) supplied through MFCs 1013 at 1 — 1000 mtorr (preferably 10 - 500 mTorr) pressure and a flow rate of 1 -100 seem. The exact dimensions of the tube and the coil, as well as the process pressure determine the spatial distribution of the plasma in the process chamber 1003. In this instrument, the power supplied to the coil can be controlled with 1 W accuracy and varies between 0 and 100 Watts. In addition, there is a tuning network that matches the impedance of the generator to the coil-tube system to maximise the input power into the plasma while keeping the reflected power low (>10%). The quartz plasma tube is sealed to the input gas manifold on one end, and to the process chamber 1003 at the other end, both using glass to metal seals. The tube and the coil are enclosed in a protective metal mesh cover. This is a typical design for generating inductively coupled plasmas of this type, although the metal to glass seals and the specific shape and outlet size have been tailored specifically for the present embodiment. The distance from the bright centre of the plasma cloud to the plane of the specimen to be etched is 20 cm, and can be varied in the range from 10 to 50 cm to adjust both the flux and the mean energy of ions reaching the surface of the specimen independent of other control parameters like stage bias.

[0118] A photograph of an embodiment of the invention is shown in Figure 10A, with the plasma generator 1002 and process chamber 1003 visible.

[0119] Within the process chamber 1003 is provided a stage 1009 with a retractable cryo shutter. The stage is connected to a temperature control means which is here a cryostat 1006, and to a voltage for application of bias to the stage.

[0120] The stage is visible in Figure 10D, a photograph of an embodiment of the invention where a shuttle with specimen is present on the stage. The image was obtained during plasma treatment, hence the clouded view.

[0121] The process chamber 1003 is connected to an exhaust 1014 via a turbo pump 1005 which acts as a pressure control means; the pump reduces the pressure in the process chamber 1003 to ultra-high vacuum levels as discussed herein. A flow restriction valve 1007 is provided to retain / maintain / control pressure conditions in the process chamber 1007. Attached to the exhaust is a mass spectrometer 1008, for analysis of material leaving the process chamber 1003. The flow restriction valve 1007, in conjunction with the MFCs 1013 for the process gas(es), are used to control the pressure in the process chamber. The use of the valve allows the desired pressure to be achieved while the turbo pump 1005 is working efficiently to minimise back streaming of gases. The turbo pump 1005 suitably has high compression ratio to minimise water content in the chamber.

[0122] Also connected to the process chamber 1003 is a load lock chamber 1010. This is here a UHV cryo transfer load deck. It is separated from the process chamber by a gate valve 1012, which ensures atmospheric separation of the load lock chamber and the process chamber when closed.

[0123] The load lock chamber 1010 is itself connectable to a specimen loader 1011 via a port, which here is controlled by a further gate valve 1012. The specimen loader 1011 may contain the specimen, for example held on a cryoEM grid in the mounting area of a preprepared shuttle, under liquid or low temperature nitrogen. It is connected to the port with the gate valve shut, and then the gate valve can be opened and the specimen brought into the load lock chamber 1010. A pressure control means for the load lock chamber (not shown) may already have reduce the pressure in the load lock chamber, or may do so at this stage, to eliminate contaminants.

[0124] The load lock chamber 1010 is visible in the upper part of Figure 10C, with the specimen holder 1011 at the bottom in a detached state.

[0125] The load lock chamber is provided with handling arms 1004. These are controllable from outside the load lock chamber. They provide a means for moving the specimen, in particular for removing it from the specimen loader into the load lock chamber and transferring it to the stage 1009 of the process chamber 1003. Here two such arms are provided.

[0126] For example, in loading, the gate valve 1012 between the load lock chamber and the process chamber is closed and that between the load lock chamber and the specimen loader is open. The handling arms 1004 are used to extract the specimen from the specimen loader 1011 , and bring it into the load lock chamber 1010. The gate valve to the specimen loader is then closed. The pressure control means (not shown) may then remove atmosphere from the load lock chamber, before the gate valve 1012 to the process chamber is opened and the specimen passed through that to the stage. The handling arm 1004 is then retracted and the gate valve 1012 shut to isolate the process chamber once more.

[0127] Alternatively, the loading may be done under an inert, water-free, for example nitrogen, atmosphere.

[0128] Loading into the load lock chamber 1010 minimises contamination using a further vacuum sealed load lock system which is pumped by a scroll pump (not shown). Loading can be done from, for example, slushed nitrogen, or from nitrogen protected environment or directly from vacuum.

[0129] The load lock chamber 1010 may be pumped by a turbomolecular pump (not shown) which can be isolated from the load lock system using a gate valve (also not shown), making it easier to recover vacuum during loading.

[0130] Optionally, for loading and unloading, the vacuum system can be vented with dry nitrogen. For unloading, the process described above can be reversed: unload from the stage 1009 using the arms 1004 into a cool pot with slushed nitrogen or liquid nitrogen, or cold nitrogen gas or vacuum.

[0131] For loading the specimen may be mounted on a shuttle (not shown), and held in a metal basket (as part of the specimen loader 1011 ) by means of a dove tail and spring loaded plate to ensure thermal contact. The loading pot 1011 is cooled with liquid nitrogen and attaches to the port below the load lock chamber 1010.

[0132] The basket is lifted into position and connected to the load lock chamber 1010, and can be left there when the specimen is carried into the process chamber 1003 by manipulation with the handling arms 1004. The handling arms may be, for example, two ultra high vacuum wobble sticks with magnetic feedthroughs, and are used to lift the basket and load the shuttle. The wobble sticks may have a thermal break at the end to avoid warming the shuttle above 120K during transfer. In the illustrated embodiment, two wobble sticks 1004 are provided to minimise the travel of the wobble sticks; each traverses a hole in the loading cryostage (and effectively has a single direction of motion, into and out of the chamber; one vertical one horizontal). The basket is lifted in position and held by a spring loaded wedge in the load lock chamber. The horizontal wobble stick is engaged onto the shuttle, by pushing against a door on the load lock chamber. The first wobble stick can be disengaged from the basket and used to open the door using an additional tool that can be attached to the end in vacuum. Mirror(s) may be mounted inside the load lock chamber to aid in attaching the wobble sticks 1004 and can be adjusted if they are on magnetic mount(s).

[0133] The load lock chamber 1010 may be cooled with a liquid nitrogen cooled cold finger, and may have sufficient mass and thermal conductivity to keep the temperature below 120K during the loading process when heating by nitrogen gases introduced during loading occurs.

[0134] The shuttle once moved to the process chamber is located on the stage 1009. The stage temperature may be controlled by feedback. Additional temperature measurement by thermocouple or thermometer may be used to monitor the temperature distribution on the stage. After plasma conditions are established, a wobble stick with low thermal mass tip, and / or tip with low thermal and electrical conductivity such as ceramic, can be used to open the shutter on the shuttle (if present). A view port may be positioned on the process chamber to allow a clear view of the shutter position through the stage and any cold trap (cryoshield) around the stage. That viewport can also be used for the optical feedback control described herein with respect to Figure 12.

[0135] The shuttle can be held in place by a spring loaded mechanism which locates the shuttle and by the dove tailed shape to ensure good thermal and electrical contact. The stage has good thermal contact to the cold head, while being electrically isolated. This may be achieved using a sapphire wafer sandwiched between two indium foils, with the stage being attached by a stainless steel bolt isolated from the stage using a ceramic washer. A protective metal shield may be provided around the cold head to protect heating coil materials from plasma impingement. Alternatively, the cold head could be totally vacuum isolated from the main chamber with a thermally conductive feedthrough.

[0136] It is possible to place a differential pumping aperture or array of apertures (mesh) between the plasma tube (ion source, 1002) and the process chamber 1003. With additional pumping on the side of the plasma tube, this can allow for lowering the pressure of non-ionised gas in the process chamber 1003, while all the ions can still be allowed to cross into the process chamber 1003 by means of applying an additional, small electrical field (in the 0 - 1000V range). Lowering the pressure of non-ionised species in the process chamber 1003 can be advantageous in reducing specimen heating during the process and increasing the mean free path for any products generated by the etching process, thereby reducing the likelihood of redeposition of etched material back onto the specimen.

[0137] Thickness Control; Patterning; Etching Rate

[0138] In methods of the present invention, the thickness of a vitrified aqueous specimen can be reduced from an initial thickness to a target (final) thickness. Of course, the target thickness is smaller than the initial thickness and the initial thickness is greater than the target thickness.

[0139] Where thickness is discussed herein, it is meant the thickness of a given part of the specimen. For example, only some parts of the specimen many have their thickness reduced: in such a case, it is the thickness of that part only which are considered as the ‘initial’ and ‘target’ thicknesses.

[0140] That is, the present methods may reduce the thickness of only a part of the specimen.

[0141] Suitably, the target thickness is a thickness appropriate to the intended use of the specimen. For example, a target thickness of <1 pm, for example 10-100 nm, may be appropriate for a specimen for cryoEM.

[0142] Similarly, the initial thickness may be chosen or depend on the nature of the specimen. For example, an initial thickness of >20 nm, for example >30 nm, >50 nm, or >100 nm, maybe be suitable to form without specimen damage in the initial freezing process.

[0143] In addition, spatial patterning and various etch profiles can be achieved by the use of masks at variable distances over the specimen.

[0144] During etching, a mask may be placed over (on one side, a to-be-etched side, of) or on (i.e. in contact with) the specimen. When not in contact with the specimen, the mask may be placed at a distance from the specimen of, for example, within one mean free path of the ions to be used at the conditions to be used. By controlling the distance between the mask and the surface, gradient profiles in milling rates can also be achieved.

[0145] The mask may have a particular design of holes and solid portions, whereby the areas of the specimen exposed by the holes in the mask will be etched and the areas concealed by the solid portions will not be etched. It is however apparent that some small amount of etching will nevertheless occur underneath the mask, close to the edges of the holes (see Figure 1 , for example).

[0146] Multiple masks of different designs may be used, either together or in sequence, to achieve a variety of patterning.

[0147] Figure 7 illustrates the use of a metal mask during low-energy plasma etching to target a selected area of the specimen for removal. Figure 7A shows the application of a rectangular metal mask 701 onto the specimen held on a cryoEM grid 702 during exposure to plasma. After etching it can be seen that only in the ‘unmasked’ area does etching proceed (etched region 703). The mask here is not in contact with the specimen but rather positioned at a distance d ‘above’ the specimen. In Figure 7B the result of experiment is shown wherein a gold grid with gold foil and amorphous ice containing lipid vesicles (liposomes) was exposed to 80 mTorr neon plasma for 23400 s with -200V bias at 65K. This resulted in the complete removal of the thin gold foil (~40 nm) and the vitreous ice in the exposed region. A composite low-magnification transmission micrograph of the grid after the exposure to the plasma is shown. Figure 7C is a high-magnification transmission electron micrograph from the region indicated with the arrow in Figure 7B and shows that in the area protected by the mask, the amorphous ice with the specimen in it and the supporting gold foil are both preserved intact.

[0148] A mask can be used to protect any arbitrary region of the specimen, allowing removal from any size and shape area.

[0149] A simple geometry using a knife edge mask at a height above the specimen foil is sufficient to provide a smooth gradient of plasma intensity across a defined area on the scale of the support, and hence a smooth gradient of thickness in the specimen. So by freezing a specimen with relatively uniform thickness across the grid there will be an area of suitable thickness for data collection. The information efficiency is greatest when the ice is thinner, containing the protein of interest. Often protein particles segregate to the air water interface and may denature there, in cases were that occurs, these surface segregated proteins can be removed leaving undamaged, randomly orientated particles. Due to avoiding interaction and alignment of particles on the air water interface, their random orientation leads to more efficient determination of structure to high resolution, more efficiently sampling the different views. To achieve this it may be necessary to make the ice sufficiently thick to lower the number of surface interactions per particle during freezing. This could be combined with methods for rapid freezing, such as jet or high pressure freezing where micrometers of ice can be frozen.

[0150] The mask geometry can be used to a create a soft gradient of exposure intensity across the grid on the scale of the path length of the gas. The mean free path of gas molecules depends on gas pressure and species (see Table 2). This can be varied from micrometer to cm. A more complex mask with multiple knife edges can be used if the path length is shorter with ice varying across each grid square rather than across the whole grid. The knife edge thickness also affects the shape of the gradient. Side walls, or other features, can also be used to provide a diffuse gradient. A sufficiently thick mask can simultaneously totally mask one well defined area of the grid, and provide a diffuse gradient across the exposed area of the grid.

[0151] Other methods could be applied to provide a soft gradient by more complex means, for example the application of shaped electromagnetic fields, application of a different electrical bias to an electrically isolated mask, or a mechanical movement of the stage or mask during the etch process.

[0152] Table 2 - Mean free paths of atoms and ions of some gases in the conditions used to generate low energy plasmas for etching vitreous specimens. For the calculation of all mean free paths, the gas is assumed to be at room temperature (293 Kelvin).

[0153] Energy dependence of etch rate and selectivity for various plasmas (refer to Figures 8 and 9)

[0154] Sputtering yield ysputterdepends on the mass difference between the incident ions and the surface atoms Maand surface binding energy of the atoms Ea.

[0155] Therefore a choice of gas with mass similar to oxygen atom or water molecule or lighter can be used, such that the plasma is more selective towards sputtering water in comparison to a metal support foil.

[0156] The binding energy of copper and gold are all expected to be about 4 eV, with metal binding energy varying from 4 to 8 eV in pure metals. Amorphous water ice (LDA, HDA) will have a binding energy less than 4 eV. Carbon and silicon also have higher binding energy but have mass closer to the oxygen.

[0157] The ion flux from the plasma onto the stage increases with applied negative voltage. Since the number of atoms sputtered by each ion (sputtering yield) also increases with ion energy, there is a cumulative effect in the rate of removal of ice with the application of negative bias: more ions arrive at the surface of the specimen per unit time and these ions impact the surface with higher energy, relative to the non-biased case. Thus, controlled application of electrical bias to the specimen can be used as a means of controlling the specimen etch rate. A power supply capable of delivering up to 1 ,000 V at constant voltage with accuracy and precision better or equal than 1 V is preferred for this application. For many applications where the selective removal of thin specimens on metal support foils is required, a voltage range of interest is between 0 V and -100 V, where the energy of the ions impacting the surface of the specimen is low enough to provide selective removal of the aqueous specimen versus the metal foil.

[0158] With a small applied voltage above 0 V there is a large increase in the current as the sheath voltage is overcome (see Figure 9). The increase in current with applied voltage depends on the gas composition and pressure. This bias can be applied to the specimen support, the whole or part of the specimen shuttle, or the specimen stage.

[0159] During plasma processing, all surfaces in contact with the plasma are susceptible to reaction and removal and must be chosen to have high stability against plasma and low outgassing in vacuum systems. Etch rate and possibility of reaction especially true on the stage or areas where the ions are accelerated by the applied bias. This can be used to lower the sputtering rate of the plasma exposed material by minimising the area with accelerating bias applied. For the stage and shuttle, a smooth surface finish with no sharp geometries also lowers the maximum field strength to lower sputtering of the stage and shuttle.

[0160] Careful consideration of the potential for redeposition of the removed material back onto other regions of the specimen must also be taken. That is, the molecules and fragments removed from the specimen need to reach another surface or be completely volatilized and pumped away with the process gasses with much higher probability than they get redeposited on another region of the specimen under etch. This can be achieved by keeping the mean free path in the gas / plasma longer than the characteristic lengths of the specimen to the specimen support (grid bars) and also the distance to any masks. Also, the nearby surfaces on the specimen support and stage should be kept at similar cryotemperatures so that any removed material landing on them does not leave the surface and end up back on the region of the specimen being etched.

[0161] Figure 8 illustrates vitreous ice sputtering rate as a function of ion energy. This graph combines data from experiments with argon and neon plasmas at various pressures and temperatures within the ranges specified in Table 1 .

[0162] Thickness monitoring / feedback control

[0163] In the present invention, etching may proceed until a desired thickness of the specimen is achieved.

[0164] While this may be approximately based on experience of etching rates under given conditions, measuring thickness during etching is desirable. Feedback from such measurements can then be used to change the etching rate (for example by modification of the applied bias), so that the desired thickness can be accurately obtained.

[0165] For example, one option is using optical monitoring of the specimen to control final thickness. To achieve optical feedback control of the specimen thickness during etching, two methods are proposed:

[0166] A. The plasma can be turned off momentarily and the specimen imaged with a light microscope setup through a window in the process chamber. This will only be limited by the optical resolution of the lenses and imaging system. It might have micrometer scale spatial resolution and thickness resolution of 100 nm using differential interference contrast or similar methods of imaging thin films.

[0167] B. A realtime feedback system is illustrated in Figure 12, based on transmitted light through the specimen. This can be achieved whilst the plasma is active by trans — illumination of the specimen with a coherent source of light at a particular wavelength (via, for example, a laser) whose amplitude is modulated at a specific frequency, and whose transmitted intensity can be measured by lock-in detection techniques even in the presence of a strong background from the light generated by the plasma.

[0168] In this implementation of the optical feedback during etching, in Figure 12, the specimen thickness is monitored via the intensity of transmitted light (I) through the specimen, which depends on the ratio of specimen thickness t to some characteristic absorbance path length, L (I = loe_(, / L), wherein Io is the intensity of incident light). To provide sufficient accuracy and precision of the measurement, the signal from the light generated by the plasma itself is filtered out, by means of wavelength filtering (matching the wavelength of the monochromated probe beam), and by lock-in detection, matching the frequency of modulation of the probe beam. If a pixelated detector is used, an image of the grid with the specimen can be formed, with resolution limited by the performance of the long working distance optics used, providing some spatial resolution (i.e. on the grid square level, ~0.1 mm) to the measurement.

[0169] In more detail, Figure 12 illustrates a variable power supply unit 1202 driving a monochrome light source 1201 . Light emitted from that passes through a viewport 1207 into the process chamber, and is reflected by a plasma-resistant mirror 1209 positioned within the process chamber such that it passes through the vitreous aqueous specimen 1210 held on a support, such as a cryoEM grid 121 1 , in the mounting area of a shuttle 1212. The transmitted light is reflected by a further plasma-resistant mirror 1209 and exits the process chamber through viewport 1207 (which may be the same viewport as that through which it entered or a different viewport). Passing through a matched wavelength filter 1206 and telescopic lens 1205 it reaches a detector 1204 which measures its intensity I. A lock-in amplifier 1203 extracts the desired signal. Through knowledge of the incident intensity Io, based on the light source, and the transmitted intensity I measured, the thickness of the specimen can be calculated by the dependency I = loe_(, / L), wherein t is the thickness of the specimen and L is the characteristic absorbance path length.

[0170] From knowledge of this thickness the operation of the processing apparatus can be adjusted, for example to reduce the bias if the desired thickness is close or to turn off if the desired thickness is reached.

[0171] ***

[0172] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof. While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0173] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0174] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0175] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0176] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0177] Reference numeral guide

[0178] 101 - vitreous aqueous specimen

[0179] 102 - etched region

[0180] 103 - interface

[0181] 104 - free diffusion zone

[0182] 105 - foil

[0183] 106 - grid bar support

[0184] 107 - mask

[0185] 108 - plasma

[0186] 701 - metal mask

[0187] 702 - grid with specimen

[0188] 703 - etched region

[0189] 1001 - apparatus / instrument

[0190] 1002 - ion source / plasma generator

[0191] 1003 - process chamber

[0192] 1004 - handling arms 1005 - pressure control means / turbo pump

[0193] 1006 - temperature control means / cryostat

[0194] 1007 - flow restriction valve

[0195] 1008 - mass spectrometer

[0196] 1009 - stage

[0197] 1010 - UH V cryo transfer load deck

[0198] 1011 - specimen loader

[0199] 1012 - gate valve

[0200] 1013 - mass flow controller(s)

[0201] 1014 - exhaust

[0202] 1201 - monochrome light source

[0203] 1202 - variable power supply unit

[0204] 1203 - lock-in amplifier

[0205] 1204 - detector

[0206] 1205 - telescopic lens

[0207] 1206 - matched wavelength filter

[0208] 1207 - UHV chamber viewport

[0209] 1208 - plasma

[0210] 1209 - plasma-resistant mirror

[0211] 1210 - vitreous aqueous specimen (thickness = t)

[0212] 1211 - specimen support / grid

[0213] 1212 - specimen shuttle

Claims

Claims:1 . A method of reducing the thickness of a vitrified aqueous specimen, the method comprising exposing the specimen to ions of energy <1000 eV while the specimen is at a temperature <150 K and in the presence of <10-6Torr of water vapour partial pressure.

2. A method according to claim 1 , wherein the ions have an energy of <500 eV, preferably <300 eV, and more preferably <100 eV.

3. A method according to claim 1 or claim 2, wherein the ions have an energy of >1 eV, preferably >2 eV, more preferably >3 eV, more preferably >4 eV, more preferably >5 eV, more preferably >10 eV.

4. A method according to any one of the preceding claims, wherein the specimen is exposed while at a temperature of <120 K.

5. A method according to any one of the preceding claims, wherein the specimen is exposed while at a temperature of >20 K.

6. A method according to any one of the preceding claims, wherein the ions are generated from at least one of helium, nitrogen, argon or neon plasma.

7. A method according to any one of the preceding claims, wherein the ions are generated from at least one of helium, nitrogen, argon plasma and at least one of oxygen or hydrogen.

8. A method according to any one of the preceding claims, wherein the specimen is exposed at a pressure of 1 mTorr to 250 mTorr, preferably 5 to 100 mTorr.

9. A method of preparing a vitrified aqueous specimen, comprising the steps of(a) mounting or forming the vitrified aqueous specimen on a stage, the specimen having an initial thickness;(b) conducting a method of reducing the thickness of the vitrified aqueous specimen according to any one of claims 1 -8, until the thickness of the specimen is reduced from the initial thickness to a target thickness which is smaller than the initial thickness.

10. A method according to claim 9, wherein the target thickness is <1 pm, preferably 10-100 nm.

11. A method according to claim 9 or claim 10, wherein the initial thickness, which is greater than the target thickness, is >20 nm, preferably >30 nm.

12. An apparatus for reducing the thickness of a vitrified aqueous specimen, comprising: an ion source which is a plasma generator or an ion beam generator;a process chamber connected to the ion source; and a stage, positioned to hold a specimen within the process chamber; a pressure control means connected to the process chamber, operable to reduce and maintain the pressure in the process chamber; and a temperature control means connected to the stage, operable to reduce the specimen temperature therein; wherein the process chamber is configured to maintain and withstand a specimen temperature of <150 K, preferably <120 K, a pressure of <250 mTorr, preferably <100 mTorr and a water vapour partial pressure of < 10-6Torr, and wherein the ion source is configured to generate ions of energy <1000 eV.

13. An apparatus according to claim 12, further comprising a shuttle for placing on the stage and having a mounting area for mounting the specimen, and a shutter on the shuttle which is moveable from a first position, in which the mounting area is exposed, and a second position, in which the mounting area is at least partially concealed.

14. An apparatus according to claim 12 or claim 13, further comprising a load lock chamber which is attached to the process chamber by an air-tight seal and separated from it by a valve, and a pump connected to the load lock chamber and operable to reduce the pressure in the load lock chamber.

15. An apparatus according to any one of claims 12 to 14, further comprising one or more gas supplies connected to the ion source, for supplying one or more processing gases to the ion source, and one or more cold traps, each corresponding to a respective gas supply and being positioned in the flow path between the gas supply and the ion source.

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